Osteoporosis treatment

ABSTRACT

There is disclosed a process of treating or alleviating the symptoms of pathological conditions in which bone density is decreased, which comprises inhibiting, in a mammalian patient suffering from such a condition, the formation in vivo of a tertiary complex of IL-11, its cell surface membrane receptor and the cell surface glycoprotein gp130. Examples of such substances are recombinant soluble IL-11 receptor mutants modified, as compared with native IL-11 receptor, at their gp130 binding site, and peptides which can interact with IL-11. The process of the invention not only inhibits bone resorption and hence bone loss, but also increases the process of bone formation to increase bone density.

RELATED APPLICATIONS

This application is a continuation-in-part of PCT applicationPCT/CA/99/00516 filed May 19, 1999, and also claims priority to Canadianapplication 2,237,915 filed May 19, 1998; the specifications of whichare incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to medical treatments and to agents usefultherein. More particularly, it relates to the prevention and treatmentof pathological conditions in which the underlying pathology is anincrease in bone resorption leading to bone loss, for examplepostmenopausal osteoporosis. It also relates to therapeutic agentsuseful in treatment and prevention of such conditions.

BACKGROUND OF THE INVENTION

The remodelling of bone depends on a balance between bone formation andbone resorption. Osteoblasts are responsible for the formation of newbone osteoid, composed mainly of nonmineralized type 1 collagen. Boneresorption is mediated by large multinucleated cells called osteoclasts.To resorb bone, osteoclasts first establish zones of dose contact withthe mineralized matrix. This forms a protected compartment between theosteoclast and the bone matrix interface in which an acidicmicroenvironment is formed. Within these zones bone is demineralized,and the collagen fibres resorbed by the action of secreted lysosomalhydrolases. A number of factors have been shown to be potent stimulatorsof bone resorption both in vitro and in vivo. These include parathyroidhormone, 1,25-dihydroxyvitamin D₃ prostaglandin-E₂ or -I₂, IL-1, TNF-α,TNF-βand bone-derived growth factors. None of these factors directlyaffect osteoclastic function; all require the presence of osteoblasts.

Increased bone resorption is a hallmark of a variety of clinicalconditions. In some common bone disorders, the balance between theprocess of resorption and formation remains normal, but the rate of boneturnover is much higher. Most cases of primary hyperparathyroidism.Paget's disease, and thyroxicosis are in this category. In other commondiseases, there is an imbalance between resorption and formation.Whether increased resorption or impaired formation predominates,however, the consequence is the same, i.e., diminished total bone mass.Thus, improper maintenance of bone occurs not only in postmenopausalwomen but is also a frequent complication of metastatic bone cancer,myeloma, and Paget's disease of bone. The present invention specificallycomtemplates that subject methods can be used as part of a treatmentprotocol for disorders marked by imbalance between resorption andformation and/or increased bone resorption.

Interleukin-11 (IL-11) has a role, either alone or in combination withother cytokines, in bone formation/resorption.

IL-11 belongs to a family of cytokines which includes interleukin-6(IL-6), leukemia inhibitory factor (LIF), and oncostatin M(OSM). Thesecytokines have similar tertiary structures, share a common signaltransducer (gp130) and have overlapping biological activities. In orderfor these cytokines to elicit a biological response, a tertiary complex,comprising the cytokine, its specific receptor (alpha chain), and gp130must be formed.

BRIEF REFERENCE TO THE PRIOR ART

Van Leuven et al., Genomics (1996) Jan 1: 31(1):65-70 report cloning thehuman gene for the interleukin-11 receptor (IL-11R), analysing thestructure of the gene, and determining the predicted protein sequence.No analysis of protein structure and function was reported.

Karow et al., Biochem J. (1996) 318: 489-495, report cloning the genefor IL-11α receptor and elucidation of its amino acid sequence, reportthe production of a soluble form of murine interleukin-11 receptor(IL-11R) and demonstrate that it interacts with the IL-11 ligand withhigh affinity. The affinity of IL-11 alone for gp130 is reported to bebelow the level of detection, but a complex of IL-11 and soluble IL-11Rinteracts with gp130 with high affinity. The receptor is a transmembraneprotein that exhibits sequence homology with other members of thehaemopoietin receptor family. However, the location of the IL-11 andgp130 binding sites on the IL-11R was not disclosed.

Teramura et al. Blood 1992, 79:327 and Musashi et al. Proc. Natl. Aced.Sci. U.S.A. 1991, 88:765 report that in bone, IL-11 functions alone orin combination with other cytokines to support granulocyte/macrophagecolony formation and to increase the number and ploidy ofplatelet-forming cells, megakaryocytes.

Girasole et al. J. Clin. Invest. 1994, 93:1516 and Tamura et al. Proc.Natl. Acad. Sc. 1993, 90:11924) report experimental work thatdemonstrates a role for IL-11 as well as other members of this family ofcytokines in the process of osteoclastogenesis.

U.S. Pat. No. 5,215,895 Bennett et al., issued Jun. 1, 1993, disclosesprocesses for production of IL-11, by culturing a cell transformed witha DNA sequence coding for IL-11.

Canadian Patent Application 2,177,837 Ciliberto et al. discloses an IL-6antagonist which has been mutated at its gp130 binding region.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel procedurefor treatment and for alleviation of the symptoms of clinicalconditions, such as osteoporosis, in which increased bone resorption ordecreased bone formation is the underlying pathology.

It is a further object of the invention to provide novel therapeuticsubstances useful in the treatment of such clinical conditions and/or inthe alleviation of symptoms thereof.

The present invention is based upon the elucidation of the role of thecytokine interleukin-11 (IL-11) in both the process of bone resorptionand the process of bone formation, and the unexpectedly advantageousresults to be obtained by inhibiting its actions. This cytokine has beenfound to be critical for osteoclast formation and activity and thereforebone resorption. Moreover, this cytokine has been found to act as aninhibitor of bone formation. The mode of action of IL-11 in its role inbone loss conditions involves the formation of a tertiary complex ofIL-11, its cell surface membrane receptor (IL-11R) and the cell surfaceglycoprotein gp130 (gp130). If this tertiary complex is not formed, oris formed to only a lesser extent, bone resorption is not onlyinhibited, but in many cases the formation of new bone is promoted, sothat effectively new bone is formed and bone density is increased.

Accordingly, the present invention from one broad aspect provides aprocess of treating or alleviating the symptoms of a pathologicalcondition in which bone density is decreased, which comprisesinhibiting, in a mammalian patient suffering from such a condition, theformation in vivo of a tertiary complex of IL-11, IL-11R and gp130. Suchconditions include those involving increased bone resorption and lack ofdesirable bone formation. In addition to osteoporosis, these conditionsinclude metastatic bone cancer, myeloma, Pagers disease of bone, andbone fracture healing, especially in the elderly human patient.

According to another aspect of the invention, there are providedtherapeutic agents for administration to a mammalian patient toalleviate the symptoms of pathological conditions in which bone densityis decreased, and comprising biologically acceptable peptide compounds,protein sequences, small molecules and antibodies capable of interferingwith the in vivo formation of a tertiary complex of IL-11, its cellsurface membrane receptor IL-11R, and the cell surface glycoproteingp130.

According to another aspect of the invention there is provided a noveluse of the TRAP and bone nodule formation assays to allow the detectionof IL-11 antagonists.

According to another aspect of the invention there are provided methodsfor the selective removal of IL-11 from a solution and methods for thepurification or enrichment of IL-11 from solutions.

According to another aspect of the invention, there are providedtherapeutic agents for administration to a mammalian patient toalleviate the symptoms of pathological conditions in which bone densityis decreased, and comprising biologically acceptable IL-11 antagonistscapable of interfering with the in vivo formation of a tertiary complexof IL-11, its cell surface membrane receptor IL-11R, and the cellsurface glycoprotein gp130.

According to another aspect of the invention, there are providedtherapeutic agents for administration to a mammalian patient toalleviate the symptoms of pathological conditions in which bone densityis decreased, and comprising transcribable genetic materials includingantisense nucleic acids capable of inhibiting the translation of acomponent necessary to the formation of the IL-11/IL-11R/gp130 tertiarycomplex.

According to another aspect of the invention, there are providedtherapeutic agents for administration to a mammalian patient toalleviate the symptoms of pathological conditions in which bone densityis decreased, and comprising expressible genetic materials includingtranscribable genetic materials encoding amino acid sequences capable ofinhibiting the formation of the IL-11/IL-11R/gp130 tertiary complex.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning ^ Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods in Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. ads.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, ads., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

BRIEF REFERENCE TO THE DRAWINGS

FIG. 1A is a diagrammatic representation of native IL-11 receptor,showing the various regions thereof and its binding interactions withIL-11 and gp130;

FIG. 1B is a representation of cDNA sequence depicted in SEQ ID NO. 3and employed in Example 3, below.

FIG. 2 is a detailed sequence of the gp130 binding region of nativeIL-11R and indicating the specifically preferred mutation sites andmutations of the products of the present invention. The extensivelymutated sequence disclosed is SEO ID NO. 4.

FIG. 3 is a representation of a portion of the IL-11 receptor peptidesequence indicating in bold the region of interaction with IL-11 (SEQ IDNO. 11).

FIG. 4 is a representation of the sequence of peptide 1 (SEQ ID NO. 1)and peptide 2 (SEQ ID NO. 2) used in Example 5 below, indicating theactivity observed in that experiment.

FIG. 5 is a graphical presentation of the results obtained according toExample 1 below.

FIGS. 6A, B and C are graphical presentations of the results obtainedaccording to Example 2 below.

FIGS. 7A and B are graphical presentations of the results obtainedaccording to Example 4 below.

FIG. 8 is a graphical presentation of the results obtained according toExample 5 below.

FIG. 9 is a graphical presentations of the results obtained according toExample 6 below.

FIG. 10 is a graphical presentation of the results obtained according toExample 7 below.

FIG. 11 is a graph showing the effect of Effect of anti-IL-11 Antibodyon osteoclast formation.

FIG. 12 is a graphical presentation of the results obtained according toExample 5 below.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The preferred process according to the invention comprises treating oralleviating the symptoms of a pathological condition in which bonedensity is decreased in a mammalian patient suffering from such acondition, by administering to the patient an effective amount of asubstance which inhibits the in vivo formation of a tertiary complex ofIL-11, its cell surface membrane receptor IL-11R and the cell surfaceglycoprotein gp130. Examples of such substances include antibodies toIL-11, antibodies to IL-11R, antibodies to gp130. mutant forms of IL-11receptor, small molecule antagonists of IL-11, and peptide compoundswhich include sequences which selectively interact with IL-11 in theregion normally bound by IL-11R, so as to interfere with the normalinteraction between IL-11 and IL-11R.

The term “IL-11 antagonist” refers to compounds which, for example,inhibit or prevent the productive interaction between IL-11 and IL-11Rand which are less effective than IL-11 at promoting the productiveinteraction of IL-11R and gp130. Prior to the observations describedherein, those skilled in the art would not have been able to predictwhether or not the surface area of interaction between IL-11 and itscognate receptor was in fact too large for a small organic molecule todisrupt. A salient feature of the observation that small peptides candisrupt IL-11 activity, e.g., by inhibiting productive interaction ofIL-11 with its receptor. Thus, the present invention specificallycontemplates that other, non-peptide small molecules can be identifiedwhich have similar abilities to disrupt IL-11/IL-11R interaction(s) andthereby function as IL-11 antagonists.

The term “antagonist” also covers agents which inhibit such intrinsicactivities of the IL-11 receptor that binding of IL-11 to its cognatereceptor produces a greatly diminished level of second messengergeneration relative to absence of treatment with the antagonist.Preferred antagonists inhibits gp130-dependent IL-11 activity.

One group of preferred compounds of the present invention arerecombinant soluble IL-11R mutants which are modified, as compared withnative IL-11R, so that they can participate in the IL-11/L-11Rinteraction but do not productively interact with gp130. As a result,the tertiary complex of IL-11, IL-11R and gp130 is not formed, or isformed only to a lesser extent, so that there is no, or a lesser,biological response. In a preferred embodiment, amino acid substitutionsin the gp130 binding region, in these preferred compounds, substantiallyor completely abolish IL-11R interactions with gp130, while havinglittle or no effect on IL-11 binding, However, mutations in otherregions of the IL-11R protein can alter the characteristics of the gp130binding site on IL-11R and prevent or inhibit productive IL-11 R/gp130interaction. Any and all soluble IL-11 receptor mutants which interferewith the formation of the IL-11/gp130/IL-11 tertiary complex, are withinthe scope of the present invention. Although the specific examplesrelate to the use of human-derived IL-11R sequences, the correspondingsequences from other mammals will also work.

The soluble IL-11Rs of the present invention are preferably mutated atone or more of positions 282, 283, 286, 289 and 291, as depicted in SEQID NO. 4, all of which are within the gp130 binding site of nativeIL-11R. Specific preferred mutations are D282 to G. A283 to D, G286 toD, H289 to Y, and V291 to L, independently or in combination of two ormore such mutations. Amino acids are described herein with reference totheir standard three letter and one letter codes. In particular, thesymbols D, G. A, H. Y, V and L have the usual meanings in connectionwith individual amino acids, namely D represents aspartic acid, Grepresents glycine, A represents alanine, H represents histidine, Yrepresents tyrosine, V represents valine and L represents leucine.

Native IL-11R is a known protein having a molecular mass of about 46 kd.Its amino acid sequence has been determined. It comprises severaldistinct functional regions, as generally illustrated in FIG. 1A of theaccompanying drawings. It is normally bound to the cell surfacemembrane. It has a region for binding to IL-11. Another of its regions,from about position 270-300, is its gp130 binding site. These regionsare indicated on FIG. 1A wherein 1 represents the amino-terminal regioncontaining 4 positional conserved cysteine residues, 2 represents anIL-11 binding region, 3 represents a gp130 binding region, and 4represents the transmembrane domain.

The amino acid sequence of the gp130 binding site is shown in FIG. 2.The present invention, in one of its preferred embodiments, providesIL-11Rs which are mutated in the gp130 binding site illustrated in FIG.2, by replacement of one or more of the native amino acids in thisregion with other amino acids. Specific preferred products of thepresent invention are those which have mutations at one or more ofpositions 282, 283, 286, 289 and 290 as illustrated and as describedabove. These mutations effectively reduce or even eliminate binding togp130, but do not materially affect binding to IL-11.

The mutant soluble IL-11Rs (sIL-11Rs) of the present invention can beprepared by known techniques. In a preferred process, cDNA encoding theIL-11R is cloned by RT-PCR using IL-11R specific primers and total RNAisolated from human osteosarcoma cells. The primers contain terminalrestriction endonuclease sites for subsequent cloning into plasmidvectors. The sequence of a preferred cDNA sequence for insertion isdepicted in FIG. 1B. After ligation into a suitable vector, the IL-11RDNA may be expressed in mammalian cells. Baby hamster kidney (BHK) cellsconstitute an example of a suitable host mammalian cell for thispurpose. After extraction and purification, the sIL-11R DNA can besubjected to site-directed mutagenesis to modify the amino acids in theIL-11 receptor which mediate gp130 binding but do not significantlyaffect IL-11 binding, for example the amino acids identified above.

A second group of preferred compounds in accordance with the presentinvention are IL-11 binding peptides, which are peptide sequences whichselectively interact with IL-11 and prevent the interaction betweenIL-11 and IL-11R necessary for the formation of the IL-11/IL-IL11R gp130tertiary complex. Surprisingly, it has been determined that small aminoacid sequences are capable of binding to IL-11 and preventing the normalinteraction between IL-11 and IL-11R. In particular, a peptide with theamino acid sequence Arg Arg Leu Arg Ala Ser Trp Thr Tyr Pro Ala Ser TrpPro Cys Gin Pro His Phe Leu (SEQ ID NO 1) has been identified whichbinds IL-11 and prevents its productive interaction with IL-11R. Evenmore surprisingly, it has been found that the peptide sequence Arg ArgLeu Arg Ala Ser Trp (SEQ ID NO 5) contained in the amino terminus of SEQID NO 1 is important to the ability of this peptide to prevent theproductive interaction of IL-11 and IL-11R. This short peptide (SEQ IDNO. 5) is also capable of inhibiting the productive interaction of IL-11and IL-11R. In addition, a third peptide (SEQ. ID. NO. 6) which islocated near, but does not overlap, the sequences corresponding to SEQ.ID. NO. 1 or SEQ. ID. NO. 5 on the human IL-11R has been found toinhibit the productive interaction between IL-11 and IL-11R.

Surprisingly, the amino acid sequences of the murine and human IL-11Rdiffer somewhat in the regions corresponding to SEQ. ID. NO 5 and SEQ.ID. NO. 6 (which depict the human sequences). In particular, the humanamino acid sequence described in SEQ. ID. NO. 5 is RRLRASW, whereas themurine sequence is RRLHASW (SEQ. ID. NO. 10). Thus, the presence of abasic amino acid residue in the position corresponding to position 4 inpeptide 1 (SEQ. ID. NO.5) is preserved between the human and murinesequences, although the actual amino acid in that position varies. Thus,the peptides corresponding to SEQ ID NO 5 and SEQ ID NO 6 are IL-11binding peptides and pepudes having the amino acid sequence RRLXASW,where X is a basic amino acid (SEQ. ID. NO.7) are potential IL-11binding peptides.

Additionally, SEQ. ID. NO. 6 depicts the amino acid sequence of an IL-11binding region identified within the human IL-11 R, namely: Ser Ile LeuArg Pro Asp Pro Pro Gin Gly Leu Arg Val Glu Ser Val Pro Gly Tyr Pro. Thecorresponding murine sequence is depicted in SEQ. ID. NO. 8 and is: SerIle Leu Arg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Val Pro Ser TyrPro. These sequences differ in their eighteenth amino acid whereby thehuman peptide has Gly and the murine sequence has Ser. Gly and Ser areboth relatively small amino acid residues, having volumes of 60.1 and89.0 Å³ respectively, and accessible surface areas of 75 and 115 Å²respectively. This suggests that the relatively small size of amino acid18 in this peptide facilitates interactions with IL-11. However, Gly andSer differ in their hydrophilicity, suggesting that several factors mayinteract to govern the suitability of particular amino acidsubstitutions at position 18 in these peptides. Thus, although IL-11binding pepUdes exist which have the amino acid sequence: Ser Ile LeuArg Pro Asp Pro Pro Gln Gly Leu Arg Val Glu Ser Val Pro xxx Tyr Pro,where xxx is a suitable amino acid, it will be necessary to screenpotential IL-11 binding peptides having this sequence using the TRAPassay and/or the bone nodule formation assay in order to determine ifthey are IL-11 binding peptides.

In addition to the amino acid substitutions discussed above, amino acidsequences from other mammals which correspond to IL-11 binding pepUdesidentified from mammalian materials will also be potential IL-11 bindingpeptides. Thus, where there is variation between mammalian sequences,having identified an IL-11 binding peptide sequence in one mammalianspecies, it is possible to identify other IL-11 binding peptides withreference to the corresponding amino acid sequences in other mammals,and the pattern of conserved residues observed.

In light of the disclosure of the present application, it is within thecapacity of a competent technician to identify and produce peptidescapable of interacting with IL-11 and inhibiting the productiveinteraction between IL-11 and IL-11R, thereby inhibiting or preventingthe formation of the IL-11/IL-11R/gp130 complex. It will be obvious toone skilled in the art that peptide sequences containing amino acidsubstitutions or modifications which preserve the features essential forbinding between the peptide and IL-11 are possible and are within thescope of the invention. Such peptides may comprise all or a portion ofthe amino acid sequence of SEQ ID NO 1, SEQ ID NO 5, SEQ. ID. NO. 6,SEQ. ID. NO.7, SEQ. ID. NO 8, or SEQ. ID. NO. 10. Alternatively, thesepeptides may have a substantially different amino acid sequence fromthese sequences, but may have functional attributes permitting specificinteractions between these peptides and IL-11. Peptides which are IL-11binding peptides may be readily dentified using one or both of the TRAPassay and the bone nodule formation ssay, discussed herein.

Protein and peptide sequences which selectively interact with IL-11 arevaluable in vitro as well as in vivo. The present invention teaches apeptide sequence which selectively binds IL-11. It is therefore wellwithin the capacity of a technician of ordinary skill to attach thispeptide to a suitable substrate by way of an appropriate linking moiety.Once attached, the immobilized peptide sequence may be used to removeIL-11 from solutions. In particular, immobilized peptides can be used todeplete solutions of IL-11. Alternatively, immobilized peptide can beused to bind IL-11 in one solution, and then release IL-11 in thepresence of a second solution, thereby allowing the production of asolution enriched in IL-11 or with a reduced number or quantity ofcomponents other than IL-11.

In other embodiments, the subject therapeutic is a peptidomimetic of anIL-11 binding peptide. Peptidomimetics are compounds based on, orderived from, peptides and proteins. The IL-11 binding peptidomimeticsof the present invention typically can be obtained by structuralmodification of a known IL-11 binding peptide sequence using unnaturalamino adds, conformational restraints, isosteric replacement, and thelike. The subject peptidomimetics constitute the continum of structuralspace between peptides and non-peptide synthetic structures; IL-11binding peptidomimetics may be useful, therefore, in delineatingpharmacophores and in helping to translate peptides into nonpeptidecompounds with the activity of the parent IL-11 binding peptides.

Moreover, as is apparent from the present disclosure, mimetopes of thesubject IL-11 binding peptides can be provided. Such peptidomimetics canhave such attributes as being non-hydrolyzable (e.g., increasedstability against proteases or other physiological conditions whichdegrade the corresponding peptide), increased specificity and/orpotency, and increased cell permeability for intracellular localizationof the peptidomimetic. For illustrative purposes, peptide analogs of thepresent invention can be generated using, for example, benzodiazepines(e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p123), C-7mimics (Huffman et al. in Peptides: Chemistry and Biologyy, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105),keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295;and Ewenson et al. in Peptides: Structure and Function (Proceedings ofthe 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill,1985), -turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231),-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126:419;and Dann et al. (1986) Biochem Biophys Res Commun 134:71),diaminoketones (Natarajan et al. (1984) Biochem Biophys Res Commun124:141), and methyleneamino-modifed (Roarketal. in Peptides: Chemistryand Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988, p134). Also, see generally, Session Ill: Analytic and syntheticmethods, in in Peptides: Chemistry and Biology, G. R. Marshall ad.,ESCOM Publisher: Leiden, Netherlands, 1988)

In addition to a variety of sidechain replacements which can be carriedout to generate the subject IL-11 binding peptidomimetics, the presentinvention specifically contemplates the use of conformationallyrestrained mimics of peptide secondary structure. Numerous surrogateshave been developed for the amide bond of peptides. Frequently exploitedsurrogates for the amide bond include the following groups (i)trans-olefins, (ii) fluoroalkene, (iii) methyleneamino, (iv)phosphonamides, and (v) sulfonamides.

Examples of Surrogates

Additionally, peptidomimietics based on more substantial modificationsof the backbone of an IL-11 binding peptide can be used. Peptidomimeticswhich fall in this category include (i) retro-inverso analogs, and (ii)N-alkyl glycine analogs (so-called peptoids).

Examples of Analogs

Furthermore, the methods of combinatorial chemistry are being brought tobear, e.g., PCT publication WO 99148897, on the development of newpeptidomimetics. For example, one embodiment of a so-called “peptidemorphing” strategy focuses on the random generation of a library ofpeptide analogs that comprise a wide range of peptide bond substitutes.

In an exemplary embodiment, the peptidomimetic can be derived as aretro-inverso analog of the peptide. Retro-inverso analogs can be madeaccording to the methods known in the art, such as that described by theSisto et al. U.S. Pat. No. 4,522,752. As a general guide, sites whichare most susceptible to proteolysis are typically altered, with lesssusceptible amide linkages being optional for mimetic switching Thefinal product, or intermediates thereof, can be purified by HPLC.

In another illustrative embodiment, the peptidomimetic can be derived asa retro-enatio analog of the peptide, such as the exemplary retro-enatiopeptide analog derived for the illustrative Arg Arg Leu Arg Ala Ser Trppeptide:NH₂-(d)Trp-(d)Ser-(d)Ala-(d)Arg-(d)Leu-(d)Arg-(d)Arg-COOH

Retro-enantio analogs such as this can be synthesized commerciallyavailable D-amino acids (or analogs thereof) and standard solid- orsolution-phase peptide-synthesis techniques. For example, In a preferredsolid-phase synthesis method, a suitably amino-protected(t-butyloxycarbonyl, Boc) D-Trp residue (or analog thereof) iscovalently bound to a solid support such as chloromethyl resin. Theresin is washed with dichloromethane (DCM), and the BOC protecting groupremoved by treatment with TFA in DCM. The resin is washed andneutralized, and the next Boc-protected D-amino acid (D-Ser) isintroduced by coupling with diisopropylcarbodiimide. The resin is againwashed, and the cycle repeated for each of the remaining amino acids inturn. When synthesis of the protected retro-enantio peptide is complete,the protecting groups are removed and the peptide cleaved from the solidsupport by treatment with hydrofluoric acid/anisole/dimethylsulfide/thioanisole. The final product is purified by HPLC to yield thepure retro-enantio analog.

In still another illustrative embodiment, trans-olefin derivatives canbe made for any of the subject polypeptides. A trans olefin analog ofIL-11 binding peptide can be synthesized according to the method of Y.K. Shue et al. (1987) Tetrahedron Letters 28:3225 and also according toother methods known in the art. It will be appreciated that variationsin the cited procedure, or other procedures available, may be necessaryaccording to the nature of the reagent used.

It is further possible couple the pseudodipeptides synthesized by theabove method to other pseudodipeptides, to make peptide analogs withseveral olefinic functionalities in place of amide functionalities. Forexample, pseudodipeptides corresponding to Ser-Ala or Arg-Leu, etc.could be made and then coupled together by standard techniques to yieldan analog of the IL-11 binding peptide which has alternating olefinicbonds between residues.

Still another class of peptidomimetic derivatives include phosphonatederivatives. The synthesis of such phosphonate derivatives can beadapted from known synthesis schemes. See, for example, Loots et al. inPeptides: Chemistry and Biology, (Escom Science Publishers, Leiden,1988, p. 118); Petrillo et al. in Peptides: Structure and Function(Proceedings of the 9th American Peptide Symposium, Pierce Chemical Co.Rockland, Ill., 1985).

Many other peptidomimetic structures are known in the art and can bereadily adapted for use in the the subject IL-11 bindingpeptidomimetics. To illustrate, the IL-11 binding peptidomimetic mayincorporate the 1-azabicyclo[4.3.0]nonane surrogate ( see Kim et al.(1997) J. Org. Chem. 62:2847), or an N-acyl piperazic acid (see Xi etal. (1998) J. Am. Chem. Soc. 120:80), or a 2-substituted piperazinemoiety as a constrained amino acid analogue (see Williams et al. (1996)J. Med. Chem. 39:1345-1348). In still other embodiments, certain aminoacid residues can be replaced with aryl and bi-aryl moieties, e.g.,monocyclic or bicyclic aromatic or heteroaromatic nucleus, or abiaromatic, aromatic-heteroaromatic, or biheteroaromatic nucleus.

The subject IL-11 binding peptidomimetics can be optimized by, e.g.,combinatorial synthesis techniques combined with high throughputscreening techniques.

Moreover, other examples of mimetopes include, but are not limited to,protein-based compounds, carbohydrate-based compounds, lipid-basedcompounds, nucleic acid-based compounds, natural organic compounds.synthetically derived organic compounds, anti-idiotypic antibodiesand/or catalytic antibodies, or fragments thereof. A mimetope can beobtained by, for example, screening libraries of natural and syntheticcompounds for compounds capable of competively inhibiting theinteraction of IL-11 and the IL-11 receptor. A mimetope can also beobtained, for example, from libraries of natural and syntheticcompounds, in particular, chemical or combinatorial libraries (i.e.,libraries of compounds that differ in sequence or size but that have thesame building blocks). A mimetope can also be obtained by, for example,rational drug design. In a rational drug design procedure, thethree-dimensional structure of a compound of the present invention canbe analyzed by, for example, nuclear magnetic resonance (NMR) or x-raycrystallography. The three-dimensional structure can then be used topredict structures of potential mimetopes by, for example, computermodelling. the predicted mimetope structures can then be produced by,for example, chemical synthesis, recombinant DNA technology, or byisolating a mimetope from a natural source (e.g., plants, animals,bacteria and fungi).

Another preferred embodiment of the present invention is a process ofselectively removing IL-11 from solutions using immobilized peptides ofthe invention having an affinity for IL-11.

Still another aspect of the invention relates to the use of non-peptidesmall molecules to antagonize IL-11 activity. The term “small molecule”as used herein refers to a compound either synthesized in the laboratoryor found in nature. Typically, a small molecule refers to an organic,i.e., carbon-containing compound, characterized in that it containsseveral carbon-carbon bonds, and has a molecular weight of less than 30kd, though more preferably less than 5000 amu, more preferably less than2500 amu and most preferably less than 1500 amu. As used herein, theterm “small molecule” may refer to short (i.e., preferably less than 20amino acid sequences) peptides, non-peptide natural products andnon-peptide compounds synthesized in the laboratory. Preferably,non-peptide compounds synthesized in the laboratory are “naturalproduct-like”, that is, possess stereochemical and functional groupdiversity as well as diversity of spatial orientation.

Particular IL-11 antagonists useful in the treatment of IL-11 relatedbone density disorders may be identified using the TRAP assay and thebone nodule formation assay, discussed herein. The TRAP assay is knownin the art and involves co-culturing murine calvarla (osteoblasts) andbone marrow cells in vitro and then quantifying osteoclast formation bycounting the number of tartrate-resistant acid phosphatase positive(TRAP+) multinucleated cells. In light of the disclosure herein, andparticularly the information pertaining to IL-11 binding peptides,including their sequences and the pattern of conserved residues, it iswithin the capacity of a competent technician to design potential IL-11antagonists which are not peptides but which have comparable bindingspecificities. These potential IL-11 antagonists may be screened todetermine if they are IL-11 antagonists by examining their activity inthe TRAP assay and the bone nodule formation assay.

Other preferred embodiments of the present invention are the use of theTRAP assay and the bone nodule formation assay, individually or incombination, to screen samples for the presence of IL-11 antagonists,and to identify IL-11 antagonists.

In preferred embodiments, the steps of the assay are repeated for avariegated library of at least 100 different test compounds, morepreferably at least 10³, 10⁴ or 10⁵ different test compounds. Exemplarycompounds which can be screened against such kekkon-mediatedinteractions include peptides, nucleic acids, carbohydrates, smallorganic molecules, and natural product extract libraries, such asisolated from animals, plants, fungus and/or microbes.

A third group of preferred compounds are peptides which bind to IL-11Rand prevent its productive interaction with IL-11. Candidate peptideswhich bind to IL-11R can be designed by molecular modelling of theIL-11/IL-11R binding site in light of the disclosure in the presentapplication. Alternatively, such peptides can be identified by screeningpotential peptides. IL-11R binding peptides may be identified frompotential peptides by the ability of IL-11R binding peptides to reducethe formation of TRAP⁺ MNC's in the TRAP assay and/or the ability ofIL-11 binding peptides to reduce the inhibitory effect of IL-11 on bonenodule formation in vitro, using the bone nodule formation assay.

As in the case of IL-11 binding peptides, the subject IL-11R bindingpeptides can also be converted into peptidomimetics, e.g., as describedabove.

In certain embodiments of the invention, an accessory peptide can beused to enhance interaction of the IL-11 derived peptide orpeptidomimetic with the target IL-11 receptor. Exemplary accessorypeptides in this regard include peptides derived from cell adhesionproteins containing the sequence “RGD”. or peptides derived from laminincontaining the sequence CDPGYIGSRC. Extracellular matrix glycoproteins,such as fibronectin and laminin, bind to cell surfaces throughreceptor-mediated processes. A tripeptide sequence, RGD, has beenidentified as necessary for binding to cell surface receptors. Thissequence is present in fibronectin, vitronectin, C3bi of complement,von-Willebrand factor, EGF receptor, transforming growth factor beta,collagen type I, lambda receptor of E. Coli, fibrinogen and Sindbis coatprotein (E. Ruoslahti, Ann. Rev. Biochem. 57:375-413, 1988). Cellsurface receptors that recognize RGD sequences have been grouped into asuperfamily of related proteins designated “integrins”. Binding of “RGDpeptides” to cell surface integrins will promote cell-surface retention,and ultimately proximity to the IL-11 receptor.

In the generation of fusion polypeptides including the subject IL-11Rbinding peptides and peptidomimetics, it may be necessary to includeunstructured linkers in order to ensure proper folding of the variouspeptide domains. Many synthetic and natural linkers are known in the artand can be adapted for use in the present invention, including the(Gly₃Ser)₄ linker.

As mentioned above, the inventive peptide compositions, including otherpeptidomimetics, non-peptide small molecules, genes and recombinantpolypeptides may be generated using combinatorial techniques usingtechniques which are available in the art for generating combinatoriallibraries of small organicipeptide libraries. See, for example,Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat.Nos. 5,359,115 and 5,362,899; the Ellman U.S. Pat. No. 5,288,514; theStill et al. PCT publication WO 94/08051; Chen et al. (1994) JACS116:2661; Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092,WO93109668 and WO91/07087; and the Lerner et al. PCT publicationWO93120242).

A fourth type of preferred compound for use in the present invention forinhibiting or preventing IL-11 activity, such as by inhibiting theformation of the IL-11/IL-11R/gp130 tertiary complex, comprisesantibodies which selectively bind to one or more of the components ofthe complex to interfere with the interaction between components to formthe tertiary complex or the intrinsic activity of the complex or subunitthereof. To illustrate, anti-IL-11 antibodies are commerciallyavailable. Alternatively, antibodies may be prepared using standardtechniques following the injection of a compound comprising the peptideof interest into a suitable animal. Useful antibodies include anti-IL-11antibodies, anti-gp130 antibodies anti-IL-11R antibodies.

The term “antibody” as used herein is intended to include wholeantibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), as well aschimeric and genetically engineered antibodies, hybrids, and fragmentsof all of the foregoing which are also specifically reactive with IL-11,IL-11R, gp130 or a complex thereof, e.g., which retain an antigenbinding site. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as for wholeantibodies. Thus, the term includes segments of proteolytically-cleavedor recombinantly-prepared portions of an antibody molecule that arecapable of selectively reacting with the target antigen. Nonlimitingexamples of such proteolytic and/or recombinant fragments include Fab,F(ab′)2, Fab′, Fv, and single chain antibodies (scFv) containing a V[L]and/or V[H] domain joined by a peptide linker. The scFv's may becovalently or non-covalently linked to form antibodies having two ormore binding sites. The subject invention includes polyclonal,monoclonal, or other purified preparations of antibodies and recombinantantibodies. In certain preferred embodiments, the subject method iscarried out using humanized monoclonal antibodies specific for a site ofinterest on one of the components of the tertiary complex.

It is also specifically contemplated that engineered protein scaffolds,e.g., for molecular recognition, can be used in place of antibodies.Suitable scaffolds in which antigen binding sites can be engineeredinclude lipocalins (c.f., Skerra (2000) Biochim Biophys Acta.1482:337-350; Beste et al. (1999) PNAS 96:1898-903; and Skerra (2000) JMol Recognit 13:167-87), and fibronectin type III domain (c.f., Koide etal. (1998) J Mol Biol. 284:1141-51).

The current invention teaches the sequence of the human IL-11R protein,the sequence of several IL-11 binding peptides, the sequence of IL-11binding regions on the human and murine IL-11Rs, and the sequence of thegp130 binding region on IL-11R, as well as the means for the productionof soluble IL-11Rs. It is, therefore, within the capability of acompetent technician to produce and screen antibodies which specificallybind either the IL-11 binding region or the gp130 binding region on theIL-11R. These specific antibodies may be used to specifically inhibitthe binding of IL-11 to the IL-11R through this binding region, therebyinhibiting formation of the aforementioned complex in vivo withoutsubstantially affecting the levels of IL-11 in solution. This may beparticularly useful in situations where it is desired to inhibit theformation of the IL-11/IL-11R/gp130 complex, but it is still desired tomaintain free IL-11 and functional gp130 to achieve some otherbiological effect.

A fifth type of preferred compound for use in the present invention forinhibiting or preventing the formation of the IL-11/IL-11R/gp130tertiary complex comprises small molecules capable of interfering withthe IL-11R/IL-11 interaction by specific binding to either IL-11 orIL-11R in the IL-11/IL-11R binding region. Small molecule antagonists ofIL-11 and IL-11R may be identified using the TRAP assay and the bonenodule formation assay. Both IL-11 antagonists and IL-11R antagonistsmay be identified and/or synthesized in light of the features of thebinding region of the compounds of the present invention. Features suchas size, shape, hydrophilicity and charge which control molecularbinding affinity are well understood, and it is well within the capacityof a competent technician to identify candidate small molecules havingthe potential to bind either IL-11R or IL-11 using molecular modellingin light of the peptides and proteins of the present invention. It isalso within the capacity of a competent technician to screen thecandidate small molecules for the ability to inhibit or prevent theformation of the IL-11/IL-11R/gp130 complex using the TRAP assay and thebone nodule formation assay, known in the prior art and described below.

In light of the teachings of the present invention, it is also withinthe capability of a competent technician to produce transcribablegenetic material capable of inhibiting the formation of theIL-11/IL-11R/gp130 tertiary complex.

One variety of transcribable genetic material which can be used is DNAencoding antisense RNA complementary to the mRNA encoding a componentnecessary to the formation of the IL-11/IL-11R/gp130 tertiary complex(including IL-11, IL-11R, gp130, or portions thereof), and capable ofinhibiting or preventing the translation of this mRNA. The mRNA sequenceof IL-11 has been previously reported and sequence listings may befounds in the GENBANK database (Accession No.s M57766, M37007 (M.Fascicularis), Accession Nos. M81890, M57765, M37006 (human)). The mRNAsequence of gp130 has been previously reported and sequence listings maybe founds the GENBANK database (Accession No.s M83336, MX62646). ThemRNA sequence of the IL-11R alpha chain has been reported and a sequencelisting may be found in the GENBANK database (Accession No. U32324). Inlight of this sequence information and the disclosure in the presentpatent application it is within the capacity of a competent technicianto produce transcribable genetic material capable of inhibiting theformation of the IL-11/IL-11R/gp130 tertiary complex. The capacity of aparticular antisense mRNA to inhibit the translation of a component ofthe IL-11/IL-11R/gp130 complex may be assessed using standard methods.

Antisense sequences may be used to inhibit the translation of thecorresponding protein of interest (including IL-11, IL-11R, and gp130),thereby effecting a reduction in levels of that protein in the treatedcells. This reduction of protein levels will reduce the proteinavailable for binding in the IL-11/IL-11R/gp130 tertiary complex,thereby reducing the formation of the tertiary complex. Transcribablegenetic material encoding antisense nucleic acid sequences may beintroduced into subjects by standard techniques, including gene therapy.

A second type of transcribable genetic material which may be used toinhibit the formation of the IL-11/IL-11R/gp130 tertiary complex istranscribable genetic material encoding amino acid sequences capable ofinhibiting the formation of the tertiary complex and containing aminoacid sequences targeting these sequences to the appropriate locationupon translation. Examples of such amino acid sequences are solublemutant IL-11Rs, IL-11 binding peptides, and IL-11R binding peptides ofthe present invention with suitable amino acid residues added whichtarget these sequences for secretion. Protein targeting and selectivecleavage sequences are known in the art and it is well within thecapacity of a competent technician to produce transcribable geneticmaterial encoding the amino acid sequence of interest and targeted forsecretion.

In addition to the amino acid sequences discussed above, it is wellwithin the capacity of a competent technician to identify other aminoacid sequences which will inhibit or prevent the formation of theIL-11/IL-11R/gp130 tertiary complex using the TRAP⁺ assay, and/or thebone nodule formation assay. Having identified a peptide or proteinwhich is capable of inhibiting the formation of the tertiary complex, itis possible to determine the amino acid sequence of that peptide orprotein using standard methods. It is therefore well within the capacityof a competent technician to devise an expressible genetic elementencoding the amino acid sequence of the peptide or protein of interestand bearing the desired post-translational modification and targetinginformation.

Transcriptional regulation elements may be selected so that thetranscribable genetic material of the present invention isconstitutively transcribed. Alternatively, the level of transcriptionmay be regulated by transcriptional control elements which are sensitiveto the level of an inducing compound. Suitable inducing compoundsinclude substances naturally produced by tissues in the patient's body,the level of which may vary with the disease state or other factors.Alternately, the inducing compound may be a substance which is notnormally present in levels sufficient to allow transcription of thetranscribable genetic material. In such a case, the inducing compoundmay be introduced into the subject at the time and dosage necessary tostimulate the desired level of transcription.

Post menopausal osteoporosis is characterized by a general reduction inbone mass, resulting from an imbalance between osteoblast-mediated boneformation and bone resorption by the osteoclast. While the osteoblast isresponsible for the formation of new bone or osteoid, it also appears tocontrol the activation and/or number of osteoclasts by releasingcytokines such as IL-6 or IL-11. This process can be examined using a“TRAP assay” which involves co-culturing murine calvaria (osteoblasts)and bone marrow cells in vitro and then quantifying osteoclast formationby counting the number of tartrate-resistant acid phosphatase positive(TRAP+) multinucleated cells. Ovariectomized (OVX) rats or mice providea satisfactory animal model for postmenopausal women in studies ofosteoporosis.

Initial experiments comparing the ability of marrow cells isolated fromsham-operated, OVX, and OVX mice treated with IL-11 neutralizingantibody, to form osteoclasts in vitro demonstrated that IL-11 Abtreatment reduces osteoclast levels below those obtained fromsham-operated animals. Accordingly, since bone density is determined bybalancing bone formation with bone resorption, inhibitors of IL-11Rbinding to gp130 reverse bone loss in post-menopausal patients.

Using oocultures of murine calvaria and bone marrow cells, it has beenshown that IL-11 is a potent stimulator of osteoclast formation invitro. Moreover, it has been demonstrated that IL-11 inhibits boneformation when murine calvaria cells (primary osteoblasts) are culturedin the presence of 250 μM ascorbic acid and 10 mM β-glycerol phosphate(the “bone nodule formation assay”). Thus, by targeting IL-11 one cannot only inhibit the process of osteoclastogenesis and thereforepathological bone loss, but one can also restore previously lost bone bystimulating the process of bone formation.

The in vitro TRAP assay also provides a convenient means to screen theeffectiveness of soluble mutant IL-11Rs, IL-11-binding peptides, IL-11Rbinding peptides, and small molecules prior to use in vivo. Inparticular, when small molecules, IL-11 binding peptides, IL-11R bindingpeptides or mutant IL-11Rs are produced, their effectiveness atinhibiting the formation of a functional tertiary complex may beassessed in vitro using the TRAP assay. Those compounds causing asignificant reduction in TRAP+ MNC's in the presence of exogenous IL-11under assay conditions are deemed to be effective at inhibiting theformation of the tertiary complex. Where a potential compound ofinterest is contained within a mixture of compounds, it may be desirableto separate these compounds by standard means prior to examination usingthe TRAP assay. Such separation will allow the removal of potentiallyundesirable compounds, as well as reducing the number of possiblecompounds producing the results observed.

In addition to allowing the identification of compounds useful toinhibit the formation of the tertiary complex, the TRAP assay allows anassessment of the relative effectiveness of various compounds of thepresent invention. The terms small molecule, IL-11 binding peptide,IL-11R binding peptide and mutant IL-11R when used herein refer to smallmolecules, peptides or proteins which are effective at inhibiting theformation of the IL-11/IL-11R/gp130 tertiary complex in vitro whentested using the TRAP+ assay. The terms anti IL-11 antibody, and antiIL-11R antibody when used herein refer to antibodies or portions orfunctional equivalents thereof which are effective at inhibiting theformation of the IL-11/IL-11R/gp130 tertiary complex in vitro whentested using the TRAP assay.

The bone nodule formation assay provides a second convenient method toscreen the effectiveness of soluble IL-11R's, IL-11 bonding peptides,IL-11R binding peptides, and small molecules at inhibiting the formationof the IL-11/IL-11R/gp130 tertiary complex. A compound to be assessedmay be added to the bone nodule formation assay system, and the effectof the compound on bone nodule formation may be assessed. As with theTRAP assay, in some circumstances it may be desirable to separatevarious compounds contained within a sample prior to examination usingthe bone nodule formation assay. Those compounds causing a significantreduction in the inhibition of bone nodule formation by IL-11 underassay conditions are deemed to be effective at inhibiting the formationof the tertiary complex. In addition to allowing the identification ofcompounds useful in inhibiting the formation of the tertiary complex,this assay allows an assessment of the relative effectiveness of variouscompounds of the present invention.

Compounds of the present invention may be administered systemically orlocally, by various modes of administration, and in various forms. Oneform in which the proteins, peptides, and small molecules of theinvention may be administered is in liquid form, as solutions orsuspensions in appropriate, biologically acceptable carriers. Apreferred means of delivering such liquid compounds is by injection.Alternately, the proteins, peptides, and small molecules of the presentinvention may be encapsulated for oral administration. The encapsulationmaterial may be selected to allow the release of the encapsulatedcompounds at an optimal stage in the digestive process to allow maximalbiological effect.

Where localized treatment is desired, the compounds of the presentinvention may be included in a suitable matrix for implantation near thedesired treatment site. Such matrices may be permanent or biodegradable,depending on the clinical needs of the patient. One particularlyeffective means of localized administration is the inclusion ofcompounds of the present invention in implanted pins used in theimmobilization of bone fractures. Another preferred form of localadministration is by injection into the region of the mammalianpatient's body proximate to the site affected by the disorder undertreatment.

The transcribable genetic material of the present invention may beadministered systemically or locally, by various modes ofadministration. Where it is desired to use antisense RNA sequences toinhibit translation of a cellular protein, the DNA encoding theantisense RNA may introduced into the nuclei of target cells by standardmeans. Where it is desired to introduce DNA sequences encoding proteinor peptide products capable of inhibiting the formation of theIL-11/IL-11R/gp130 tertiary complex, the DNA may be introduced into thenuclei of cells already present in the patient's body by standard means.Alternatively, the DNA may be introduced into the nuclei ofMHC-compatible cells by standard means in vitro, and the resulting cellsexpressing the introduced DNA may be administered to the patientsystemically or locally by standard means such as intravenous injection,or local injection near sites of concern.

The desired dosage of active compound will vary, depending on the modeof administration, the condition to be treated, the overall condition ofthe subject, and the compound administered. It is well within thecapability of a competent technician to determine the appropriate dosagefor a particular patient in light of these factors. It is anticipatedthat where the systemic administration of the solubilized mutant lL-11Rof the present invention by injection is desired, the appropriate dosagewill be between 1 mg to 20 mg of the soluble mutant IL-11R per kg bodyweight. Depending on the subject and the condition to be treated,dosages will be more preferably between 1 to 10 mg per kg body weightfor subjects whose existing bone density is not extremely low andbetween 10 mg to 20 mg per kg body weight for subjects whose bonedensity is extremely low. It is anticipated that in many pathologicalconditions the appropriate systemic dosage of solubilized mutant IL-11Rwill be similar to the appropriate dosage of parathyroid hormone for thetreatment of a comparable patient.

Where localized administration of the compounds of the present inventionis desired, the appropriate localized dosage can be determined withreference to the level of compound desired in the treatment area. It isanticipated that the total dosage required for localized treatment willbe lower than that level required for systemic treatment, and in manycases the appropriate localized dosage will be ten to one-hundred foldlower than the amount of compound required for systemic treatment.

Where it is desired to use a small molecule, IL-11 binding peptide,IL-11R binding peptide or a specific antibody instead of, or in additionto, solubilized mutant IL-11R, the appropriate dosage may be easilydetermined based on the appropriate dosage of IL-11R. The smallmolecule, peptide or antibody dosage will be a function of thesolubilized mutant IL-11R dosage, adjusted to provide a comparable levelof effective target binding based on the abundance of the target, therelative molecular mass and binding affinity of the small molecule,peptide or antibody for its target relative to the solubilized mutantIL-11R, its relative effectiveness at blocking tertiary complexformation in vitro, and its relative half-life In vivo. Targetabundance, molecular mass, binding affinity and in vivo half life of aparticular small molecule, antibody or peptide may be determined bystandard methods. The effectiveness of the compound at blocking theformation of the tertiary complex in vitro may be assessed using theTRAP assay and/or the bone nodule formation assay, as previouslydiscussed. It is anticipated that the appropriate dosage of theIL-11-binding peptides of the present invention when administered bylocal injection will frequently be between 0.1 to 10 mg per kg bodyweight. The dosage of a particular IL-11 binding peptide needed for aparticular patient may be easily determined with reference to thefactors discussed above, the patient's overall condition, and thedisorder to be treated.

In some instances it will be desirable to enhance the activity and/or invivo half-life of the peptides of the present invention by the chemicalmodification of these peptides to increase activity or to inhibit invivo degradation. For example, chemical moieties may be covalentlyattached to specific amino acids to interfere with the action ofdegradative enzymes. Alternatively or additionally, specific amino acidsin the peptide sequence may be chemically modified to increase theoverall peptide half life without greatly reducing specific bindingbetween the peptide and IL-11. Additionally, it will be desirable insome instances to employ one or more D-isomer amino acid residues in theformation of these peptides. In some instances it will be desirable tocyclize the peptides. For example, cyclization can be used to stabilizea particular peptide conformation in order to obtain a desired level ofbinding. Methods of modifying peptide sequences by the addition ofchemical moieties to amino acids, the chemical modification of specificamino acids, the cyclization of peptides, and the incorporation ofD-isomers of amino adds are known in the art and it is within thecapacity of a competent technician in light of this disclosure todetermine what modifications are appropriate and how to effect thesemodifications.

Where the compound to be administered comprises antisense geneticmaterial to be expressed within the patient's cells, the appropriatedose will also depend on the level of transcription of the antisensematerial, and its stability and binding affinity for its complementaryIL-11R RNA in the cell.

Attentively or additionally, expressible genetic material encoding asecreted form of a protein or peptide sequence of interest may beintroduced into suitable MHC-compatible cells ex vivo by standardmethods and these cells may be introduced into the body of the patientby standard methods. These cells may be administered locally in theregion where increased bone deposition or decreased bone resorption isneeded, or they may be administered generally. The expressible geneticelement may be constitutively active, or it may be inducible. Theappropriate dosage of expressible genetic elements will depend on themode of administration, the condition to be treated, the patent'scondition, the stability of the coding RNA and its protein product, thelevel of protein expression, and other factors influencing the level ofeffective target binding, as discussed in relation to IL-11 bindingpeptides, IL-11R binding peptides, small molecules and specificantibodies. In general, it is anticipated that the appropriate dosage ofexpressible genetic elements will be the dosage which leads to a levelof secreted protein or peptide in the vicinity of the target cells whichis generally similar to the level of protein or peptide present in thevicinity of the target cells when the appropriate level of that peptideis administered directly to the patient.

Where the compound to be administered comprises an antibody whichspecifically binds to IL-11, IL-11R. or gp130 and prevents or inhibitsthe formation of the tertiary complex, either systemic or localizedadministration of the compound may be possible. Localized administrationmay be accomplished by various means, including the injection of asolution containing the antibody of interest into the region proximateto the tissue to be treated. Alternatively or additionally,MHC-compatible cells secreting the antibody of interest may beintroduced into the subject's body in either a systemic or a localizedmanner.

The invention is further described, for illustrative purposes, in thefollowing specific examples.

EXAMPLE 1 IL-11 Inhibits Bone Nodule Formation

The effect of IL-11 on bone formation was examined using the bone noduleformation assay. Murine calvaria cells (primary osteoblasts) werecultured in the presence of 250 μM ascorbic add and 10 mM β-glycerolphosphate, and in the presence of various amounts of interleukin-11.After culturing, the culture media were plated out and the number ofbone nodules per plate was counted. The results are shown on theaccompanying FIG. 5, where number of bone nodules per plate is plottedas the vertical axis, with the different concentrations of interleukinon the horizontal axis, not to scale. Each bar represents the result onexperiments containing a different concentration of interleukin-11, asnoted on FIG. 5.

It is clearly seen that the experiment conducted in the absence ofinterleukin-11 led to the highest number of bone nodules per plate, andthat the number of bone nodules per plate decreased as the interleukin-11 concentration increased. This demonstrates that IL-11 inhibits boneformation.

EXAMPLE 2 Demonstration of the Ability of Anti-IL-11-NeutralizingAntibodies to Halt and to Reverse Bone Loss in Ovariectomized Animals

Twenty four laboratory mice were divided into four equal groups of six.Three of the groups were ovariectomized (OVX) while the fourth group wassham-operated (SHAM), to act as a control. One week afterovariectomization, treatment of one group of ovariectomized animals(OVX+Anti IL-11 Ab) with a daily dose of 160 μg/mouse of anti-IL-11 Ab(an affinity-purified sheep anti-murine IL-11 polyclonal neutralizingantibody) commenced. At the same time, treatment of another group ofovariectomized animals (OVX+NSIgG) with the same daily dosage of normalsheep immunoglobulin (NSIgG) was commenced. Treatments were deliveredonce daily by intraperitoneal injection. Plasma analysis demonstratedthat IgG entered the circulation of NSIgG group animals. Animals in theremaining two groups received no treatment.

On the day on which treatment was commenced, the sham-operated animals(Sham-Baseline) and an equal number of the untreated ovariectomizedanimals (OVX-Baseline) were sacrificed to obtain their right femurs forhistomorphometric analysis, so that baseline values could beestablished.

On day 21 after the commencement of the treatment, the remaining animalswere similarly killed and their right femurs removed for bonehistomorphometry. For this, the undecalcified distal third of the rightfemur of each mouse was embedded in glycolmethacrylate (JB-4 embeddingmedium; Analychem, Toronto, Ontario, Canada). Histologic sections of 6to 8 μm were obtained using a Riechert Jung microtome (model K4;Riechert Jung Canada, Toronto, Ontario), mounted, and then stained witheither 1% toluidine blue or hematoxyline and eosin (H & E) before beingsubjected to morphometric analysis. In each case, a region 800 μm belowthe epiphyseal growth plate that included the entire metaphysis wassubjected to light microscopy using a Merz grid (Carl Zeiss Canada, DonMills, Ontario). Sections examined in this fashion encompassed a totaltissue area of 5-8 mm². The following parameters were determined: (1)cancellous bone volume, (2) osteoblast surface, (3) osteoid surface, and(4) osteoclast surface. For each section, cancellous bone volume wascalculated from a total of >1,600 point measurements (45 fields; 400×magnification), which were selected at random using the Merz grid. Thepercent osteoblast, osteoid, or osteoclast surface was calculated underoil immersion (1,000×) by recording the presence or absence of eachwhere the hemispherical grid of the Merz radical crossed cancellousbone. Osteoblasts were identified morphologically as distinctcuboidal-shaped cells lining the cancellous bone surface, whereasosteoclasts were identified morphologically as large multinucleatedcells in close proximity to the cancellous bone surface, which stainedfor tartrate-resistant acid phosphatase (Sigma Chemical Co., St. Louis,Mo.; Procedure No. 386).

The results of measurements of cancellous bone volume are presentedgraphically in FIG. 6A. Clearly the animals of the OVX+Anti IL-11 Abgroup, treated with IL-11 antibody, had a much larger volume ofcancellous bone than the untreated OVX group and the negative controlOVX NSIgG, IgG treated group. Measurements on the femurs from sham andOVX animals sacrificed on the day on which treatment was commencedestablish baseline values for the bone volume increases. It will benoted that the test animals of the OVX+Anti IL-11 Ab group showed asignificant gain in cancellous bone volume as compared to OVX baseline,indicating that cancellous bone loss was not only prevented but that itwas reversed by the inhibition of biological activity of IL-11.

FIG. 6B of the accompanying drawings presents the results of osteoidsurface measurements, and indicates that the OVX+Anti IL-11 Ab animalsexhibited higher rates of bone formation than the comparative groups,demonstrating that by inhibiting IL-11 biological activity one canpromote the formation of new bone, reverse bone loss, and increase bonedensity in OVX mice.

FIG. 6C of the accompanying drawings similarly presents the results ofosteoclast surface measurements, and shows a notable reduction in thecase of the OVX+Anti IL-11 Ab animals treated with the IL-11 antibodies.This again indicates that the OVX+Ant IL-11 Ab animals exhibited muchless bone resorption than the comparative groups, in furtherdemonstration of the fact that inhibition of biological activity ofIL-11 prevents and even reverses bone resorption in OVX mice.

EXAMPLE 3 Preparation of Soluble Interleukin-11 Receptor That InhibitsIL-11 Induced Osteoclast Formation

cDNA encoding the IL-11 receptor (minus the transmembrane andcytoplasmic domains) was cloned by RT-PCR using IL-11 receptor specificprimers and total RNA isolated from the human osteosarcoma cell lineSAOS-2. Primer sequences were based on the DNA sequence for the humanIL-11 receptor α-chain. The forward primer contains a Kozak consensussequence preceding the start ATG codon, while the reverse primercontains, in addition to a termination codon, bases encoding a histidinetag. Both primers contain terminal restriction endonuclease sites forsubsequent cloning into plasmid vectors. Authenticity of the cDNA insertencoding the soluble IL-11 receptor (cDNA depicted in SEQ ID NO 3) isconfirmed by restriction endonuclease analysis and by double-strandedDNA sequencing using a modified T7 DNA polymerase system (Sequenase,Amersham).

For stable expression in mammalian cells, the IL-11R cDNA isgel-purified and ligated into the pcDNA3.1 vector(Invitrogen, Carlsbad,Calif.) which encodes a neomycin gene and allows for selection underhigh concentrations of G418. The cDNA is inserted upstream of the humancytomegalovirus (CMV) immediate-eariy promoter/enhancer (this allows forhigh-level expression in a variety of mammalian cell lines) anddownstream of the bovine growth hormone (BGH) polyadenylation signal(which allows for efficient transcript stabilization and termination).The cDNA sequence to be inserted was formed from the cDNA sequencecorresponding to nucleotides 62 to 1156 on the IL-11R cDNA as identifiedby Van Leuven et al. with an additional 39 nucleotides added to the 3′end of this sequence to provide a thrombin cleavage site, a histidinetag, and a stop codon. The cDNA sequence inserted (SEQ ID NO. 3) isdepicted in FIG. 1B.

Proper orientation of the inserted IL-11R cDNA is confirmed by usingrestriction endonuclease analysis and DNA sequence analysis. Afterapproximately 10-12 days of selection in medium containing neomycin,drug-resistant colonies are isolated and the highest secreting clonesare seeded into roller bottles and the medium collected every 2-3 days.Soluble IL-11R is purified from the collected medium using a Ni²⁺-IDAcolumn and subsequently eluted using EDTA. Antigen levels are determinedby ELISA using antibodies to both the histidine tag as well as the IL-11receptor.

To assure that the sIL-11 R does not associate with gp130, site-directedmutagenesis is used to modify the amino acids in the IL-11 receptorwhich mediate gp130 binding but do not affect IL-11 binding.Specifically, the process mutates D282 to G, A283 to D, G286 to D, H289to Y, and V290 to L, independently or in combination. The relevantportion of the IL-11R, and the preferred mutations, are depicted in FIG.2. Site-directed mutagenesis is performed as described previously(Austin, Richard C. et al. “FEBS Letters”, Vol. 280, No. 2, 254:258(March 1991) Federation of European Biochemical Societies) using mutantoligodeoxynucleotide primers synthesized at the Central Facility of theinstitute for Molecular Biology and Biotechnology, McMaster University.Prior to expression in BHK cells, the resultant sIL-11R mutant cDNAs arethen inserted into pcDNA3.1, and the sequences of all the sIL-11R mutantcDNAs are confirmed by sequencing as described above.

EXAMPLE 4 Determination of the Effect of A sIL-11R Antagonist on IL-11Induced Osteoclast Formation in vitro

The effect of IL-11 on osteoclast development in cocultures of murinebone marrow and calvaria cells was examined using standard techniques.Briefly, bone marrow cultures were established by removing femurs frommice, dissecting away soft tissue, and removing the distal and proximalends of the femur. The marrow was then flushed with 5 ml α MEM and 1.0%penicilin-streptomycin using a 25 gauge needle. The bone marrow cellswere then suspended to a concentration of 5,000,000 cells per ml in αMEM containing 15% fetal calf serum (charcoal treated) to remove cellsadherent to plastic. The non-adherent cells were then co cultured for anadditional 9 days with murine calvaria cells prior to being fixed andstained for TRAPase activity (stains were obtained from Sigma ChemicalCo., St. Louis, Mo.). TRAP+MNC's have the ability to form resorptionpits in smooth cortical bone slices and are therefore considered to beof osteoclast origin.

(i) IL-11 Dose-dependancy of TRAP+Cell Formation

The effect of IL-11 on osteoclast development in cocultures of murinebone marrow and calvaria cells was examined by maintaining thesecultures in the presence of various specific concentrations of IL-11 for9 days. After 9 days of culture, the cells were stained for TRAPaseactivity and the number of multinucleated TRAP+cells were determined.The results of this experiment are depicted in Table I. Data areexpressed as mean +/−SEM.

(ii) Impact of Mutant IL-11 Receptor on IL-11 Induced OsteoclastFormation

In order to assess the effect of a gp130 binding region mutant IL-11R onIL-11 induced osteoclast formation, the procedure described in (i) abovewas repeated using 20 ng/ml IL-11 and 10,100, or 1000 ng/ml of either(A) the H289→Y289 mutant solubilized IL-11 receptor described in Example3, or (B) a corresponding solubilized native IL-11 receptor. The resultsof this experiment, depicted in FIGS. 7A and B, demonstrate that amutant IL-11 receptor is capable of inhibiting IL-11-induced osteoclastformation, whereas the native IL-11 receptor is not.

EXAMPLE 5 Determination of the Effect of A Peptide IL-11R Antagonist onIL11 Induced Osteoclast Formation in vitro

It is desirable to have a means of selectively inhibiting theinteraction of IL-11 with the IL-11R, without adding exogenousantibodies or other large proteins. Surprisingly, it was determined thata short peptide sequence could be created which is capable of inhibitingthe interaction between IL-11 and the IL-11R. A peptide with the aminoacid sequence Arg Arg Leu Arg Ala Ser Trp Thr Tyr Pro Ala Ser Trp ProCys Gln Pro His Phe Leu (“Opeptide 1”, SEQ ID NO. 1) was synthesizedwhich is homologous to a region in the IL-11 receptor which appears tobind IL-11.

To determine if peptide 1 could inhibit IL-11-induced MNC formation, theprocedure of Experiment 4 was repeated using peptide 1 in place of thegp130 binding region mutant IL-11 receptor protein, and using anoverlapping peptide (“peptide 2”, SEQ ID NO. 2) in place of thesolubilized native IL-11 receptor. Peptide 2 has the amino acid sequenceThr Tyr Pro Ala Ser Trp Pro Cys Gin Pro His Phe Leu Leu Lys Phe Arg LeuGln Tyr (SEQ ID NO 2) and represents a portion of the amino acidsequence occurring in the native IL-11 receptor, and overlapping in partwith the sequence of peptide 1. Peptide 2 lacks the N-terminal Arg ArgLeu Arg Ala Ser Trp sequence (SEQ ID NO. 5) which is contained inpeptide 1. The sequence of these peptides is depicted in FIG. 4.

Peptide 1 inhibits IL-11 induced osteoclast formation, whereas peptide 2does not. The results of this experiment pertaining to peptide 1 aredepicted in FIG. 8. This indicates that a peptide sequence comprisingArg Arg Leu Arg Ala Ser Trp is capable of interacting with IL-11 andacting as an antagonist to IL-11 mediated activation of the osteoclastformation. The ability of peptide 1, and particularly the peptidesequence Arg Arg Leu Arg Ala Ser Trp to inhibit osteoclast formationindicates that this peptide is interacting with IL-11. Thus, peptide 1is an example of an IL-11 binding peptide.

FIG. 12 shows the ability of the peptide Ser Ile Leu Arg Pro Asp Pro ProGln Gly Leu Arg Val Glu Ser Val Pro Gly Tyr Pro to IL-11 inducedosteoclast formation.

EXAMPLE 6 Determination of the Effect of IL-11 Antagonists on TheAbility of IL-11 to Inhibit Bone Nodule Formation in vitro

It was desired to determine if the IL-11 antagonists of the inventioncould control bone nodule formation, and in particular, if theseantagonists could reduce the inhibitory effect of IL-11 on bone noduleformation.

Bone nodule formation was measured using standard techniques. Briefly,calvaria cell cultures were established as follows: Calvaria cells wereobtained from 2 day old fetal mouse calavariae by collagenase digestion.The cells were then cultured for 21 days in the presence of 0.5 mMascorbic acid and 10 mM β-glycerophosphate. Where indicated in theexperimental descriptions below, IL-11 and the mutant IL-11 receptor ofexperiment 4 were added to the culture at day 0 and every 3-4 daysthereafter until the removal of the medium for analysis. Bone noduleformation was quantified by counting alizarin red stain nodules under alight microscope.

Exogenous IL-11 alone added to murine calvaria cell culture can inhibitbone nodule formation, as depicted in FIG. 5. The effect of a gp130binding region mutant soluble IL-11 receptor of type described inExperiment 4 and produced by the process of Experiment 3 on bone noduleformation in the presence of 20 ng/ml of exogenous IL-11 was assessed,and the results are depicted in FIG. 9. These results indicate that verylow levels of mutant solubilized IL-11 receptor can reverse the effectof exogenous IL-11. Moreover, the addition of 10 ng/ml of the mutantreceptor used in this experiment was capable of allowing enhanced bonenodule formation relative to the level observed in the absence ofexogenous IL-11. Thus, IL-11 antagonists such as mutant IL-11Rs canenhance bone nodule formation.

EXAMPLE 7 Determination of the Effect of an IL-11 Antagonist Peptide onthe Ability of IL-11 to Inhibit Bone Nodule Formation in vitro

It was desired to determine if bone nodule formation could be modulatedin a manner similar to that reported in Example 6 using an IL-11antagonist peptide such as the one used in Example 5. To achieve this,the procedure of Example 6 was repeated, with the substitution ofpeptide 1 for the solubilized mutant IL-11 receptor. The results of thisexperiment are depicted in FIG. 10. Peptide 1 was able to reduce theinhibitory effects of 20 ng/ml exogenous IL-11 at very lowconcentrations, and at concentrations of 50 ng/ml, peptide 1 allowedbone nodule formation in excess of that seen in the control samples.Peptide 2 was unable to reduce the inhibition of nodule formation byIL-11. Thus, peptide 1 (SEQ ID NO.1), and particularly the peptidesequence Arg Arg Leu Arg Ala Ser Trp (SEQ ID NO.5), is capable ofallowing enhanced bone nodule formation.

EXAMPLE 8 Effect of Anti-IL-11 Antibody on Osteoclast Formation

Using an osteoclast formation assay, treatment with anti-IL-11 antibodyis shown to inhibit of osteoclast formation and activity inovarectomized mice. Briefly, bone marrow cells are isolated from fromovariectromized mice by flushing mouse femur with 5 ml α-MEM. The marrowcells were cultured for 9 days in α-MEM containing 10% FCS and 10 nMVitD₃ and then stained for osteoclasts by detecting tartrate-resistantacid phosphastase activity. Osteoclasts were quatitated using lightmicroscopy. As FIG. 11 illustrates, the number of TRAP+ multinucleatedcells (osteoclasts) I reduced by treatment of the animals withanti-IL-11 antibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, publications and patents cited in the specification areherein incorporated by reference.

1. A composition of matter comprising a mutant IL-11R (Interleukin-11Receptor) mutated in the gp130 binding site of a wild-type IL-11R, andwherein said mutant IL-11R is an IL-11 (Interleukin-11) signalingantagonist.
 2. The composition of claim 1, in which the mutant IL-11Rhas at least one of the following mutations: D282→G282, A283→D283,G286→D286, H289→Y289, and V291→L291.
 3. The composition of claim 2 inwhich the mutant IL-11R has the H289→Y289 mutation.
 4. The compositionof claim 1, wherein the mutant IL-11R is soluble.
 5. The composition ofclaim 1, wherein the mutant IL-11R is a human IL-11R.